Genetics: Early Online, published on July 27, 2016 as 10.1534/genetics.116.189217 Accurate chromosome segregation at first meiotic division requires AGO4, a protein involved in RNA-dependent DNA methylation in Arabidopsis thaliana Cecilia Oliver1, Juan Luis Santos and Mónica Pradillo Departamento de Genética, Facultad de Biología, Universidad Complutense de Madrid, Spain 28040. 1 Present address: Institute of Human Genetics, UPR 1142 CNRS, Montpellier, France 34396. 1 Copyright 2016. Running title: AGO4 role during Arabidopsis meiosis Key words: AGO4 Arabidopsis thaliana Centromere Meiosis RdDM Corresponding author: Mónica Pradillo Mailing address: Departamento de Genética, Facultad de Biología, Universidad Complutense de Madrid, José Antonio Nováis 12, Madrid, Spain 28040. Phone number: +34 913944764 Email address: pradillo@bio.ucm.es 2 ABSTRACT RNA-directed DNA methylation (RdDM) pathway is important for the transcriptional repression of transposable elements and for heterochromatin formation. Small RNAs are key players in this process throughout a feedback with both DNA and histone methylation. Taking into account that methylation underlies gene silencing and that there are genes with meiosis-specific expression profiles, we have wondered whether genes involved in RdDM could play a role during this specialized cell division. To address this issue we have characterized meiosis progression in pollen mother cells (PMCs) from Arabidopsis thaliana mutant plants defective for several proteins related to RdDM. The most relevant results were obtained for ago4-1. In this mutant, meiocytes display a slight reduction in chiasma frequency, alterations in chromatin conformation around centromeric regions, lagging chromosomes at anaphase I, and defects in spindle organization. These abnormalities lead to the formation of polyads instead of tetrads at the end of meiosis, and might be responsible for the fertility defects observed in this mutant. Findings reported here highlight an involvement of AGO4 during meiosis by ensuring accurate chromosome segregation at anaphase I. 3 INTRODUCTION RNA-dependent DNA methylation (RdDM) confers transcriptional repression in all sequence contexts (Matzke et al. 2009; Law and Jacobsen 2010). In this specialized RNAi pathway, the base-pairing between 24 nt small interfering RNAs (siRNAs) and nascent scaffold transcripts directs the DNA methylation complex to target loci (Law and Jacobsen 2010; Matzke and Mosher, 2014). In Arabidopsis thaliana, the two catalytic subunits of RNA polymerase IV (Pol IV), NRPD1A and NRPD1B, generate single strand RNAs (ssRNAs) which serve as templates for RNA-dependent RNA polymerase 2 (RDR2) to produce double strand RNAs (dsRNAs). These dsRNAs are subsequently cleaved by DICER-LIKE3 (DCL3) into 24 nt siRNAs, exported to the cytoplasm and loaded onto AGO4, AGO6, or AGO9 (Matzke et al. 2009; Law and Jacobsen, 2010; Zhang and Zhu, 2011; Pikaard et al. 2012; Ye et al. 2012). Afterwards, these complexes are imported back to the nucleus to target transcripts generated by Pol V at the same loci, before they are released from the chromatin (Wierzbicki et al. 2008, 2009; Liu et al. 2014). Additionally to the sequence complementarity between the 24 nt siRNAs and the nascent Pol V transcripts, Pol V subunit NRPE1 possesses an AGO4binding motif (known as the AGO hook), located in the carboxy-terminal region (ElShami et al. 2007). AGO4 is then able to recruit the DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to establish de novo DNA methylation (for a detailed characterization of this pathway see Bologna and Voinnet 2014; Borges and Martienssen 2015). RdDM affects transcription of transposons and repeated DNA elements through de novo methylation of cytosines in all sequence contexts (CG, CHG and CHH 4 contexts, where H denotes A, T, or C). In this DNA methylation the methyltransferases DRM1 and DRM2 plays a key role (Cao and Jacobsen 2002a; Henderson et al. 2010; Law and Jacobsen 2010). However, other methyltransferases, such as CHROMOMETHYLTRANSFERASE 3 (CMT3) and DNA METHYLTRANSFERASE 1 (MET1) are involved in CHG and CG methylation maintenance, respectively (Jones et al. 2001; Cao and Jacobsen 2002b; Cao et al., 2003). Furthermore, CHG methylation, previously established by DRM2, is recognized by the H3K9 histone methyltransferase KRYPTONITE (KYP), reinforcing the repressed chromatin state of methylated DNA (Cao et al. 2003; Sasaki et al. 2012). On these grounds, we have wondered whether genes involved in siRNA biogenesis and RdDM could be important during meiotic division in A. thaliana. In fission yeast and animals, the regulation of histone methylation is necessary for meiotic recombination (Wahls et al. 2008; Acquaviva et al. 2013; Crichton et al. 2014). In maize, the absence of proteins involved in DNA methylation leads to alterations in sporogenesis and megagametogenesis (García-Aguilar et al. 2010). In this species, defective mutants for AGO104 (ortholog of AGO9) display an apomixis-like phenotype, revealing that this gene is essential during meiosis (Singh et al. 2011). In A. thaliana, it has been reported that methylation status might influence meiotic homologous recombination (HR). The hypomethylated ddm1 (decrease in DNA methylation 1) and met1 mutant plants display a total number of reciprocal genetic exchanges, crossovers (COs), similar to wild-type (WT) plants. However, they show differences in the recombination frequency along chromosomes respect to WT plants (Melamed-Bessudo and Levy, 2012; Mirouze et al., 2012; Yelina et al., 2012). Additionally, plants defective for AGO9, a protein from the same phylogenetic clade than AGO4 which is 5 expressed in the germ-line, show a slight increase in the mean chiasma frequency per cell respect to WT plants (Oliver et al. 2014). Here we present results that reveal the influence of a protein required for RdDM, AGO4, on chromatin organization at both centromeric and pericentromeric regions, highlighting its importance in ensuring an accurate chromosome segregation. MATERIALS AND METHODS Plant materials All the mutants evaluated are in Col background except ago4-1 which is in Ler (Zilberman et al. 2003). ago4-2 is a dominant mutation resulting from the substitution of a Glu at position 641 (located inside the PIWI domain) by a Lys (Agorio and Vera 2007). Mutant seeds were kindly donated by Dr. Pablo Vera (Universidad de Valencia, Spain). The remaining mutants correspond to T-DNA insertion lines and they were obtained from Salk Institute Genomic Analysis Laboratory and provided by the Nottingham Arabidopsis Stock Centre (NASC) (Alonso et al. 2003). In this work, we have analyzed the following single mutants: ago4-1, ago4-2, ago4-1t, ago6-2, dcl3-1, kyp-4, nrpd1a-8, and nrpe1-11; and the triple mutants: cmt3-11 drm1-2 drm2-2, met1-3 drm1-2 drm2-2, and kyp-6 drm1-2 drm2-2. Plants were cultivated on a soil mixture of vermiculite and commercial soil (3:1) and grown in a greenhouse under a 16/8 hours light/dark photoperiod, at 18-20 with 70% humidity. Primers listed in Table S1 and LBb1.3 (5´ATTTTGCCGATTTCGGAAC3´, for SALK lines) or LB2 (5´GCTTCCTATTATATCTTCCCAAATTACCAATACA3´for SAIL lines) were used for genotyping. 6 Seeds from Ler and ago4-1 (n = 215 and n = 218, respectively) were put on plates containing GM medium to assess the germination percentage. The number of germinated seeds in each background was evaluated 9, 11, 13, and 18 days after sowing. Cytological analysis Pollen viability was quantified by Alexander staining (1969) with some modifications (Peterson et al. 2010). Fixation of flower buds, slide preparations and fluorescence in situ hybridization (FISH) were performed according to Sánchez-Morán et al. (2001). The following DNA probes were used: pTa71 (45S rDNA; Gerlach and Bedbrook 1979), pCT4.2 (5S rDNA; Campell et al. 1992), pAL1 (centromeric DNA repeat, Martínez-Zapater et al. 1986), and pLT11 (telomeric DNA repeat, Richards and Ausubel 1988). Chromosome preparations for subsequent immunolocalizations of histone H3 modifications, centromeric histone H3 variant (CENH3), and α-tubulin were obtained by a squash technique as described by Manzanero et al. (2000), with minor modifications (Oliver et al. 2013). ASY1, ZYP1, RAD51 and DMC1 proteins were immunodetected according to the spreading protocol described by Armstrong et al. (2002) (Table S2). Secondary antibodies were FITC conjugated (1:50, Sigma) and Cy3 conjugated (1:300, Sigma). Immunolocalization of 5mC was previously described in Oliver et al. (2014). Sensitivity to -rays Seeds from Ler and ago4-1 were surface sterilized in 2.5% sodium hypochlorite solution during five minutes. After three washes in sterile water and an overnight at 4°, the seeds were exposed to 150, 300, and 500 Gy (2.94 Gy/min) from a 7 137 Cs source (IBL 437C CIS BIO International) and sown on GM plates. The number of true leaves and the fresh weight of the seedlings were recorded 14 days after treatment. qPCR Expression analyses were performed as previously described by Pradillo et al. (2012). Details of the primers and probes used are shown in Table S3. Fold variation was considered over a calibrator using the ΔΔCt method (Livak and Schmittgen 2001). Statistical analyses Statistical analyses used in this work were managed with the software SPSS Statistics 17.0. Table S1 contains the sequence of the primers used for genotyping. Information about the antibodies used is provided in Table S2. Table S3 contains the sequence of the primers used in the expression analyses and numbers corresponding to the TaqMan probes (Roche). RESULTS Plant fertility and seed germination in mutants of genes related to RdDM We have analyzed the following single mutants of genes related to RdDM: ago4-1 (knockout, KO; Ler background), ago4-1t, ago4-2 (knockdown, KD; Col background), ago6-2, dcl3-1, kyp-4, nrpd1a-8 (defective for one of the catalytic subunits of Pol IV), 8 nrpe1-11 (defective for the largest subunit in Pol V). We have also examined triple mutants defective for different methyltransferases: cmt3-11 drm1-2 drm2-2, met1-3 drm1-2 drm2-2, and kyp-6 drm1-2 drm2-2. Only the single mutant ago4-1 and the triple mutant met1-3 drm1-2 drm2-2 showed reduced fertility. The semisterility of the triple mutant may be explained by defects in gametogenesis and embryo-viability (Saze et al. 2003; Xiao et al. 2006). However, in ago4-1 we detected interplant variation in the number of non-viable pollen grains (2-21%), suggestive of meiotic defects (five flowers analyzed; Figure S1), and also a reduction in the percentage of germinated seeds respect to WT plants: 46.38% vs. 98.13% (18 days after sowing; t = 14.77; p < 1 x 10-3; Figure S2). Furthermore, a proportion of ago4-1 seeds (20.4%) showed smaller size and dehydrated appearance, 2.3% presented two root apical meristems (RAMs), and 0.9 % did not show any RAM (n = 216, Figure S2). Cytological analysis of meiosis Pollen mother cells (PMCs) from ago4-1 displayed the highest number of meiotic alterations among all mutants analyzed, namely: i) A different chromatin compaction at centromeric and pericentromeric regions at pachytene (Figures 1A, 1C). In WT PMCs there are two strongly DAPI stained regions per bivalent corresponding to pericentromeric regions that flank the centromere, which is faintly DAPI stained (Figure 1B). However, in ago4-1 centromeric and pericentromeric regions were not clearly distinguished because they showed a similar DAPI staining intensity (Figure 1D); ii) Chromosome decondensation from diplotene to metaphase I (Figures 1G, 1H, 1K). 47% of meiocytes at metaphase I (n = 121) showed this feature (Table 1). In addition, some bivalents displayed centromeric regions with forked appearance (Figure 1L); iii) 9 Presence of interchromosomal bridges (Figure 1O), lagging chromosomes (Figure 1P), and occasional fragments at anaphase I (Figures 1S, 1T). The percentage of these cells was around 30% (n=26). However, this percentage could be overestimated, since defects on chromosome segregation might increase the duration of this stage; iv) Presence of chromatin accumulations and isolated chromosomes at late telophase II. These defects are responsible for the formation of polyads in 47.7 % of cells analyzed (n = 300; Figures 1W, 1X). The mutant ago4-2 showed meiotic alterations similar to those described in ago4-1, although in minor extent: slight differences in the conformation of centromeric and pericentromeric regions at pachytene respect to WT; chromosome decondensation from diplotene to metaphase I, exhibiting 27% of decondensed metaphases I (n = 177; Table 1; Figures 1I, 1M); interchromosomal bridges and lagging chromosomes at anaphase I that originate the abnormalities observed at metaphase II (Figures 1Q, 1U); and 3% of polyads (n = 300; Figure 1Y). Female meiosis was apparently normal in both ago4 mutants (Figure S3), but the results are not conclusive due to the low number of cells analyzed. The mutants dcl3-1, ago6-2, nrpe1-11, kyp-4, and kyp-6 drm1-2 drm2-2 also displayed chromatin decondensation in a percentage of diplotene-metaphase I PMCs (Table 1; Figure S4). Interchromosomal anaphase I bridges were observed in ago6-2, kyp-6 drm1-2 drm2-2, and kyp-4 (Figures S4F-S4H), but, in contrast to ago4-1 and ago4-2, chromosome fragments and isolated chromosomes were absent. Finally, meiosis seems to be cytologically normal in nrpd1a-8, ago4-1t (mutant with the T-DNA insertion into the promoter), cmt3-11 drm1-2 drm2-2, and met1-3 drm1-2 drm2-2. 10 Chiasmata were scored according to previously established criteria (SánchezMorán et al. 2001, 2002). Between one and three plants per mutant were analyzed to estimate mean cell chiasma frequencies at metaphase I. Only three mutants showed significant differences in this parameter respect to WT plants: ago4-1 and ago4-2 exhibited a significant decrease (t = 2.92; p = 4 x 10-3, and t = 3.11; p = 2 x 10-3, respectively), whereas nrpd1a-8 presented a significant increase (t = 2.44; p = 16 x 103 ). The remaining mutants analyzed displayed a general tendency toward a slight, although non-significant, increase of this parameter (Table 2). At chromosome level, there was a significant increase of chiasma frequency in the short arms of chromosomes 2 (nrpd1a-8, kyp-4, ago6-2, met1-3 drm1-2 drm2-2, dcl3-1, and ago4-2) and 4 (nrpd1a8, ago6-2, dcl3-1, and ago4-2). By contrast, ago4-1 and ago4-2 showed a decrease in chiasma frequency in both chromosome 1 and the long arm of chromosome 2. Regarding chromosome 3, there was a significant reduction in the short arm (ago4-1) and in the long arm (ago6-2, nrpe1-11, cmt3-11 drm1-2 drm2-2, and ago4-2). Finally, met1-3 drm1-2 drm2-2, ago4-2, and nrpe1-11 displayed a decrease in the long arm of chromosome 5. Since the most relevant results obtained from a meiotic point of view are those referred to ago4 mutants, particularly ago4-1, we decided to perform a more accurate study based on the following issues: i) characterization of centromeric and pericentromeric regions; ii) histone modifications during meiosis; iii) synapsis and HR; and iv) chromosome segregation. Additionally, we have analyzed possible defects in mitosis and DNA repair. Characterization of centromeric and pericentromeric regions 11 As mentioned before, centromeric and pericentromeric regions of ago4-1 and ago4-2 displayed a different DAPI staining respect to WT, especially at pachytene. To gain insight into this phenomenon, we performed a FISH using the centromeric sequence of 180 bp (pAL1), and a telomeric probe (PLT11) as a positive control. The overall size of the centromeric signals was conspicuously smaller in the ago4 mutants than in WT plants, while no apparent differences in the size of the telomeric signals were observed (Figure 2). We also detected differences among the chromosomes. We performed an immunodetection of 5-methyl cytosine (5mC), because heterochromatic regions in plant chromosomes are usually enriched in this DNA modification. In Col, Ler and ago4-2, 5mC was restricted to pericentromeric regions (Figures 3A-3D), but in ago4-1 it was located at both pericentromeric and centromeric regions (Figures 3E-3H). Additionally, both mutants displayed a normal distribution of CENH3, even in meiocytes with abnormal chromosome segregations (Figures S5-S7). Analysis of histone modifications during meiosis Dimethylated H3K9 (H3K9me2) is the main histone H3 modification regulated by RdDM and it is located at heterochromatic pericentromeric regions. We did not detect any variation in the distribution of this histone modification between ago4 and WT plants (Figure S8). The distribution was also similar for the following euchromatic H3 modifications (Oliver et al. 2013): H3K4me2, H3 acetylated (H3Ac) and H3K27me3 (Figures S9-S11). We have also analyzed the chromosome distribution of H3S10Ph, a modification associated with condensation in mammals (Cobb et al. 1999) and with the process of sister chromatid cohesion in plants (Kaszas and Cande 2000; Manzanero et al. 2000). In A. thaliana, this post-translational modification appears at diplotene- 12 diakinesis and remains until the beginning of anaphase I. It reappears again at metaphase II to be finally absent at anaphase II (Oliver et al. 2013). This pattern was broadly similar in ago4-1, ago4-2 and WT pachytene PMCs (Figure 4; Figure S12), although a slight delay in H3S10Ph disappearance was observed at anaphase I in ago4-1 (Figure 4). This delay was probably associated to chromosome regions involved in interchromosomal bridges (Figures 4A-4L). The pattern of appearance-disappearance at second meiotic division was similar to that observed in WT plants (Figures 4M-4T). Synapsis and homologous recombination (HR) Since ago4-1 and ago4-2 were the only mutants that showed a significant reduction in the mean cell chiasma frequency as compared to WT plants, we decided to analyze HR and synapsis by means of the immunolocalization of different proteins. The recombinases RAD51 and DMC1 were loaded normally onto meiotic axes and synapsis progressed correctly according to the detection of ASY1 and ZYP1, proteins associated to the lateral and central elements of the synaptonemal complex (SC), respectively (Figure S13). In ago4-1 we have also analyzed the expression of some representative genes involved in HR: SPO11-1, ATM, ATR, BRCA1, BRCA2B, RAD50, RAD51, RAD51C, DMC1, MSH4, MLH3, MUS81, SMC6A, and SMC6B. In bud samples (enriched in meiocytes) only SPO11-1 (2.52-fold), and SMC6B (2.36) were over-expressed, while DCM1 (4.43), and MUS81 (2.11) only were in leaf samples (Figure S14). ATR (0.40), RAD51C (0.35), and SMC6B (0.33) were under-expressed in leaf samples. Meiotic chromosome segregation 13 To assess whether abnormalities in chromosome segregation at anaphase I observed in ago4-1 were related to alterations in the structure and/or function of the spindle, we examined this structure by α-tubulin immunolocalization. The most intriguing finding was the presence of microtubule bundles with an altered disposition, located in transversal orientation with respect to the division axes at anaphases I and II (Figure 5). The immunodetection of α-tubulin in the polyads revealed that some of the four pollen grains originated were multinucleate (Figure S15). However, we did not detect abnormal mature pollen grains with more than three nuclei. Hence, it is feasible to think that irregular pollen grains observed might degenerate before the occurrence of pollen mitoses (Figure S16). Mitosis and sensitivity to γ-rays Simultaneously to meiotic characterization, a cytological analysis of mitosis in ago4-1 and ago4-2 was conducted. Prophase was apparently normal, but we observed associations between decondensed chromosomes at metaphase (36%; n = 50), anaphases with delay chromatid segregations (36% in ago4-1, 32% in ago4-2; n = 50) and interchromatid bridges (5% in both mutants; n = 50). However, telophases were apparently normal (Figure 6). Due to the presence of these mitotic defects we decided to evaluate possible deficiencies in DNA repair by irradiating seeds of ago4-1 and Ler with γ-rays. This DNA damaging agent has a high mutagenic potential in plants and produces a large number of lesions which mainly generate double-strand breaks (DSBs). Among the different doses of irradiation, only at 450 Gy the mutant showed a significant decrease 14 respect to WT plants in both the number of leaves and the fresh weight per plant (t = 2.53; p = 13 x 10-3; Figure S17). DISCUSSION The semisterility displayed by ago4-1 had only been associated to developmental floral organ defects (Zilberman et al. 2004). However, our results suggest that it can also be related to the formation of unviable pollen grains (Figure S1). Furthermore, ago4-1 seeds showed a germination delay (Figure S2) and a considerable variability at morphological level, similar to that observed in superman (sup) mutants, defective for a zinc finger protein (Gaiser et al. 1995). Indeed, AGO4 is involved in silencing the SUP gene (Zilberman et al. 2003). Centromeric and pericentromeric regions Among all mutants analyzed, the most conspicuous meiotic alterations were observed in ago4 mutants (Figure 1), especially in ago4-1 PMCs. Observations reported here suggest that differences in chromatin organization at both centromeric and pericentromeric regions respect to WT could play a role in this scenario (Figures 2 and 3). The main components of Arabidopsis centromere are a sequence of 180 bp in thousands of tandemly repeated copies and transposons like Athila, Tat, Tim or Copia. Only 15% of 180 bp sequences are connected to CENH3 (Nagaki et al. 2003; Schubert et al. 2012). In contrast to the centromere, pericentromeric regions can show different condensation levels (Schubert et al. 2012). These regions have a low gene density and are rich in transposons and repeated DNA sequences (May et al. 2005). In ago4-1 the 15 signal size corresponding to the 180 bp sequence observed by FISH was considerably smaller than in the WT (Figure 2). This reduction in DNA FISH signals could reflect a partial centromeric deletion, although changes in chromatin conformation could also difficulty the accessibility of the probe to the complementary DNA sequence. However, apparent changes in the location pattern of CENH3, required to kinetochore assembly, were not detected (Figures S5-S7). Zilberman et al. (2003) reported that ago4-1 does not affect methylation levels at the 180 bp sequence, but we have observed a decrease in 5mC at pericentromeric regions, associated with a punctate pattern at centromeres (Figure 3). This anomalous 5mC distribution could be related to the alterations detected during anaphase I (Figures 1, 3 and 5). Mutations in fission yeast of genes coding for proteins involved in siRNA pathway produce abnormalities in chromosome segregation, and also a loss of pericentromeric heterochromatin silencing detected by a decrease in the signal corresponding to H3K9me2 (Volpe et al. 2003; Fukagawa et al. 2004; Kanellopoulou et al. 2005). However, we have not detected dissimilarities in the distribution pattern of this and others H3 modifications in ago4 mutants (Figures S8S11). Chromatin architecture The final effect of RdDM is methylation of DNA that is determinant in chromatin compaction (Liu et al. 2016). Indeed, siRNAs are involved in the recruitment of chromatin modification complexes that lead to the formation of heterochromatin (Wassenegger, 2005; Matzke and Mosher, 2014). General chromatin decondensation observed from diplotene to metaphase I was a common feature in the mutants of genes involved in RdDM, although the percentage of decondensed nuclei was variable among 16 them. The highest percentages corresponded to ago4-1 and nrpe1-11 (Table 1; Figures 1 and S4). However, we did not observe differences between ago4-1 and WT PMCs in the distribution pattern of H3Ac, despite histone deacetylase 6, HDA6, is necessary for the propagation of de novo methylation directed by RNA (Aufsatz et al. 2002). Likewise, we did not detect differences for other H3 post-translational modifications (Figures S8, S9 and S11). These results concur with those reported in other mutants that show alterations in the siRNA machinery (Naumann et al. 2005; Pontes et al. 2009). Homologous recombination Drosophila mutants defective in piRNA/ra-siRNA components do not show important changes in CO frequency (Cross and Simmons, 2008). In A. thaliana, we have found only three mutants for genes involved in RdDM with significant variations in the mean cell chiasma frequency respect to WT plants: ago4-1 and ago4-2 showed a decrease while nrpd1-8 displayed an increase (Table 2). The results obtained here also reveal that the same bivalent, and their chromosome arms, may behave in a different way in different mutants (Table 2). This indicates the existence of factors controlling meiotic recombination at arm/chromosome level that are differentially regulated in the mutants. In this sense, met1 and ddm1 mutants present a reduction in DNA methylation at heterochromatic pericentromeric regions and changes in CO frequency along chromosomes, although their overall chiasma frequency is similar to that observed in WT plants (Melamed-Bessudo and Levy, 2012; Mirouze et al. 2012; Yelina et al. 2012). Maintenance of chromosome architecture is important for the correct function of meiotic proteins. Nevertheless, and despite the reduction in CO frequency, ago4-1 and 17 ago4-2 showed full synapsis and the recombinases RAD51 and DMC1 were normally loaded onto chromatin (Figure S13). Furthermore, expression analysis of genes involved in meiotic HR did not reveal any clear tendency (Figure S14). Additionally, ATR and RAD51C and SMC6B are under-expressed in the somatic line of ago4-1 (Figure S14), despite the mutant is not hypersensitive to γ-rays (Figure S17). Similar results have been found in other mutants affected in siRNA biogenesis in which there is a low spontaneous HR frequency and DNA repair enzymes are not overexpressed (Wei et al. 2012; Yao et al. 2016). Chromosome segregation ago4 mutants exhibited some abnormalities at mitotic anaphases, although telophases were normal (Figure 6). They also displayed defects during meiosis, especially at anaphase I, lying in the presence of interchromosomal bridges and a delay in chromosome segregation (Figure 1). These abnormalities were also observed, although in minor extension, in nrpe1-11, kyp-4 and kyp-6 drm1-2 drm2-2 (Figure S4). Chromosome bridges at anaphase I have been described in mutants defective in the repair of programmed DSBs. In these mutants, the bridges are mostly accompanied by chromosome fragments that are detected at late prophase I (Schommer et al. 2003; Puizina et al. 2004; Wang et al. 2012). However, in ago4-1 chromosome fragmentation was almost nominal and restricted to anaphase I (Figures 1 and 4). In A. thaliana H3 phosphorylation occurs all along the chromosomes from diplotene to telophase I, and from late prophase II to metaphase II, disappearing at telophase II (Caperta et al. 2008; Oliver et al. 2013; Figure S12). Manzanero et al. (2000) and Kaszas and Cande (2000) have suggested that in plants H3 phosphorylation 18 is related to sister chromatid cohesion. Thus, it is tempting to speculate that the association of H3S10ph to bridges and chromosome fragments at anaphase I could reflect the difficulties in the separation of sister chromatids (Figure 4). Other actors in this scenario could be members of the Aurora family (Demidov et al. 2005) that are responsible for the cell-cycle dependent phosphorylation of H3 at serine 10 (Demidov et al. 2009), and for ensuring correct chromosome segregation (Demidov et al. 2014). In addition, depletion of Aurora B in Drosophila, Caenorhabditis elegans and results in reduced H3S10ph and is related to defects in chromosome segregation (Wei et al. 1999; Adams et al. 2001; Giet and Glover 2001). Also, a mutation of H3S10 in Tetrahymena produces segregation defects (Wei et al. 1999). In Arabidopsis meiosis, the alterations in the dynamics of this modification observed in ago4-1 could be responsible for the improper spindle formation (Figure 5). Alterations in chromatin condensation, failures in spindle morphogenesis and polyad formation were also observed in the ago104 maize mutant, and in the Arabidopsis mutants radially swollen 4 (rsw4) and tardy asynchronous meiosis 1 (tam1), defective for a separase and a cyclin, respectively. These mutants also show delayed chromosome segregation (d´Erfuth et al. 2010; Singh et al. 2011; Yang et al. 2011). This phenomenon, although less extreme, is also displayed by defective mutants for CENH3 (Ravi et al. 2010; Lermontova et al. 2011). In mammals, defects in spindle formation and chromosome alignment have also been detected in the female meiosis of mutants defective for endogenous siRNAs (Stein et al. 2015). In conclusion, the changes in the organization of centromeric and pericentromeric chromatin observed in ago4 are probably the origin of the failures observed in spindle organization. Alterations in kinetochore-microtubule interactions 19 could be responsible for chromosome segregation defects, manifested cytologically as chromosome bridges and fragments. Cytokinesis at the end of the meiotic process would lead to the formation of four meiotic aberrant products. Altogether these results suggest that AGO4 may possess a meiosis-associated cellular function that seems to be independent of other proteins also involved in RdDM. In this sense, recent publications have highlighted the existence of siRNAs generated via an alternative route independent of DCLs (siRNAs independent of DCLs, sidRNAs). These sidRNAs are associated with AGO4 and capable of directing DNA methylation, playing key roles in the initiation of RdDM (Ye et al. 2015). Additionally, although in general AGO6 is redundant with AGO4 in RdDM, they can play non-redundant roles in regulating the same RdDM target and may act sequentially to mediate siRNA-guided DNA methylation (Duan et al. 2015). It could explain the differences in the mutant phenotypes that we have analyzed. Although further studies should be needed to decipher the detailed mechanism how AGO4 influences Arabidopsis meiosis, this study marks the way. ACKNOWLEDGMENTS This work has been supported by grants from European Union Framework Program 7 (Meiosys-KBBE-2009-222883) and Ministerio de Economía y Competitividad of Spain (AGL2012-38852). We thank Dr. Tomás Naranjo (Universidad Complutense de Madrid, Spain), Dr. Andreas Houben (IPK, Germany), and Dr. Chris Franklin (University of Birmingham, UK) for providing antibodies used in this work. ago4-2 seeds were kindly donated by Dr. Pablo Vera (Universidad de Valencia, Spain). 20 REFERENCES Acquaviva, L., Székvölgyi, L., Dichtl, B. S., de la Roche Saint André C., Nicolas, A. et al., 2013 The COMPASS subunit Spp1 links histone methylation to initiation of meiotic recombination. Science 339: 215-218. Adams, R. R., Maiato, H., Earnshaw, W. C., Carmena, M., 2001 Essential roles of Drosophila inner centromere protein (INCENP) and Aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. J. Cell Biol. 153: 865-880. Agorio A., and Vera P., 2007. ARGONAUTE4 is required for resistance to Pseudomonas syringae in Arabidopsis. Plant Cell 19: 3778-3790. Alexander, M. P., 1969 Differential staining of aborted and nonaborted pollen. Stain Technol. 44: 117-122. Alonso, J. M., Stepanova, A. N., Leisse, T. J., Kim, C. J., Chen, H. et al., 2003 Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653-657. Armstrong, S. J., Caryl, A., Jones, G., and Franklin, F., 2002 Asy1, a protein required for meiotic chromosome synapsis, localizes to axis-associated chromatin in Arabidopsis and Brassica. J. Cell Sci. 115: 3645-3655. Aufsatz, W., Mette, M. F., Van der Winden, J., Matzke, M., and Matzke, A. J. M., 2002 HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA. EMBO J. 21: 6832-6841. Bologna, N. G., and Voinnet, O., 2014 The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Ann. Rev. Plant Biol. 65: 473-503. 21 Borges, F., and Martienssen, R. A., 2015 The expanding world of small RNAs in plants. Nat. Rev. Mol. Cell Biol. 16: 727-741. Campell B. R., Song Y., Posch T. E., Cullis C. A., and Town C. D., 1992 Sequence and organization of 5S ribosomal RNA-encoding genes of Arabidopsis thaliana. Gene 112: 225-228. Cao, X., and Jacobsen, S. E., 2002a Role of the Arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing. Curr. Biol. 12: 1138-1144. Cao, X. and Jacobsen, S. E., 2002b Locus-specific control of asymmetric and CpNpG methylation by the DRM and the CMT3 methyltransferase genes. Proc. Natl. Acad. Sci. USA 99: 16491-16498. Cao, X., Aufsatz, W., Zilberman, D., Mette, M. F., Huang, M. S. et al., 2003 Role of the DRM and CMT3 methyltransferases in RNA-directed DNA methylation. Curr. Biol. 13: 2212-2217. Caperta, A. D., Rosa, M., Delgado, M., Karimi, R., Demidov, D. et al., 2008 Distribution patterns of phosphorylated Thr 3 and Thr 32 of histone H3 in plant mitosis and meiosis. Cytogenet. Genome Res. 122: 73-79. Cobb, J., Miyaike, M., Kikuchi, A., and Handel, M. A., 1999 Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase IIα localization and chromosome condensation. Chromosoma 108: 412-425. Crichton, J. H., Playfoot C. J., and Adams, I. R., 2014 The role of chromatin modifications through mouse meiotic prophase. J. Genet. Genomics 41: 97-106. Cross, E. W., and Simmons, M. J., 2008 Does RNA interference influence meiotic crossing over in Drosophila melanogaster? Genet. Res. 99: 253-258. 22 d’Erfurth, I., Cromer, L., Jolivet, S., Girard, C., Horlow, C. et al., 2010 The cyclin-A CYCA1;2/TAM is required for the meiosis I to meiosis II transition and cooperates with OSD1 for the prophase to first meiotic division transition. PLoS Genet. 6: e1000989. Demidov, D., Van Damme, D., Geelen, D., Blattner, F. R., and Houben, A., 2005 Identification and dynamics of two classes of aurora-like kinases in Arabidopsis and other plants. Plant Cell 17: 836-848. Demidov, D., Hesse, S., Tewes, A., Rutten, T., Fuchs, J. et al., 2009. Aurora1 phosphorylation activity on histone H3 and its cross-talk with other post-translational histone modifications in Arabidopsis. Plant J. 59: 221-230. Demidov, D., Lermontova, I., Weiss, O., Fuchs, J., Rutten, T. et al., 2014. Altered expression of Aurora kinases in Arabidopsis results in aneu-and polyloidization. Plant J. 80: 449-461. Duan, C. G., Zhang, H., Tang, K., Zhu, X., Qian, W. et al., 2015. Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA-directed DNA methylation. EMBO J. 34: 581-592. El-Shami, M., Pontier, D., Lahmy, S., Braun, L., Picart, C. et al., 2007 Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components. Genes Dev. 21: 2539-2544. Fukagawa, T., Nogami, M., Yoshikawa, M., Ikeno, M., Okazaki, T. et al., 2004 Dicer is essential for formation of the heterochromatin structure in vertebrate cells. Nat. Cell Biol. 6: 784-791. Gaiser, J. C., Robinson-Beers, K., and Gasser, C. S., 1995 The Arabidopsis SUPERMAN gene mediates asymmetric growth of the outer integument of ovules. Plant Cell 7: 333-345. 23 García-Aguilar, M., Michaud, C., Leblanc, O., and Grimanelli, D., 2010 Inactivation of a DNA methylation pathway in maize reproductive organs results in apomixis-like phenotypes. Plant Cell 22: 3249-3267. Gerlach, W. L., and Bedbrook, J. R., 1979 Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res. 7: 1869-1885. Giet, R., and Glover, D. M., 2001 Drosophila Aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J. Cell Biol. 152: 669-682. Henderson, I. R., Deleris, A., Wong, W., Zhong, X., Chin, H. G. et al., 2010 The de novo cytosine methyltransferase DRM2 requires intact UBA domains and a catalytically mutated paralog DRM3 during RNA-directed DNA methylation in Arabidopsis thaliana. PLoS Genet. 6: e1001182. Jones, L., Ratcliff, F., and Baulcombe, D. C., 2001 RNA-directed transcriptional gene silencing in plants can be inhherited independently of the RNA trigger and requires MET1 for maintenance. Curr. Biol. 11: 747-757. Kanellopoulou, C., Muljo, S. A, Kung, A. L., Ganesan, S., Drapkin, R. et al., 2005 Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev. 19: 489-501. Kaszás, E., and Cande, W. Z., 2000 Phosphorylation of histone H3 is correlated with changes in the maintenance of sister chromatid cohesion during meiosis in maize, rather than the condensation of the chromatin. J. Cell Sci. 113: 3217-3226. Law, J. A., and Jacobsen, S. E., 2010 Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11: 204-220. 24 Lermontova, I., Koroleva, O., Rutten, T., Fuchs, J., Schubert, V. et al., 2011 Knockdown of CENH3 in Arabidopsis reduces mitotic divisions and causes sterility by disturbed meiotic chromosome segregation. Plant J. 68: 40-50. Liu, Z. W., Shao, C. R., Zhang, C. J., Zhuo, J. X., Zhang, S. W. et al., 2014 The SET domain proteins SUVH2 and SUVH9 are required for Pol V occupancy at RNAdirected DNA methylation loci. PLoS Genet. 10: e1003948. Liu, Z. W., Zhou, J. X., Huang, H. W., Li, Y. Q., Shao, C.R. et al., 2016 Two components of the RNA-Directed DNA methylation pathway associate with MORC6 and silence loci targeted by MORC6 in Arabidopsis. PLoS Genet. 12: e1006026. Livak, K. J., and Schmittgen, T. D., 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402-408. Manzanero, S., Arana, P., and Puertas, M. J., 2000 The chromosomal distribution of phosphorylated histone H3 differs between plants and animals at meiosis. Chromosoma 109: 308-317. Martínez-Zapater, J. M., Estelle, M. A., and Somerville, C., 1986 A highly repeated DNA sequence in Arabidopsis thaliana. Mol. Gen. Genet. 204: 417-423. Matzke, M., Kanno, T., Daxinger, L., Huettel, B., and Matzke, A. J., 2009 RNAmediated chromatin-based silencing in plants. Curr. Opin. Cell Biol. 21: 367-376. Matzke, M. A., and Mosher, R. A., 2014 RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat. Rev. Genet. 15: 394-408. 25 May, B. P., Lippman, Z. B., Fang, Y., Spector, D. L., and Martienssen, R. A., 2005 Differential regulation of strand-specific transcripts from Arabidopsis centromeric satellite repeats. PLoS Genet. 1: e79. Melamed-Bessudo, C., and Levy, A. A., 2012 Deficiency in DNA methylation increases meiotic crossover rates in euchromatic but not in heterochromatic regions in Arabidopsis. Proc. Natl. Acad. Sci. USA 109: E981-988. Mirouze, M., Lieberman-Lazarovich, M., Aversano, R., Bucher, E., Nicolet, J. et al., 2012 Loss of DNA methylation affects the recombination landscape in Arabidopsis. Proc. Natl. Acad. Sci. USA 109: 5880-5885. Nagaki, K., Talbert, P. B., Zhong, C. X., Dawe, R. K., Henikoff, S. et al., 2003 Chromatin immunoprecipitation reveals that the 180-bp satellite repeat is the key functional DNA element of Arabidopsis thaliana centromeres. Genetics 163: 12211225. Naumann, K., Fischer, A., Hofmann, I., Krauss, V., Phalke, S. et al., 2005 Pivotal role of AtSUVH2 in heterochromatic histone methylation and gene silencing in Arabidopsis. EMBO J. 24: 1418-1429. Oliver, C., Pradillo, M., Corredor, E., and Cuñado, N., 2013 The dynamics of histone H3 modifications is species-specific in plant meiosis. Planta 238: 23-33. Oliver, C., Santos, J. L., and Pradillo, M., 2014 On the role of some ARGONAUTE proteins in meiosis and DNA repair in Arabidopsis thaliana. Front. Plant Sci. 5: 177. Peterson, R., Slovin, J. P., and Chen, C., 2010 A simplified method for differential staining of aborted and non-aborted pollen grains. Int. J. Plant Biol. 1: e13. 26 Pikaard, C. S., Haag, J. R., Pontes, O. M., Blevins, T., and Cocklin, R., 2012 A transcription fork model for Pol IV and Pol V-dependent RNA-directed DNA methylation. Cold Spring Harb. Symp. Quant. Biol. 77: 205-212. Pontes, O., Costa-Nunes, P., Vithayathil, P., and Pikaard, C. S., 2009 RNA polymerase V functions in Arabidopsis interphase heterochromatin organization independently of the 24-nt siRNA-directed DNA methylation pathway. Mol. Plant 2: 700-710. Pradillo, M., López, E., Linacero, R., Romero, C., Cuñado, N. et al. 2012 Together yes, but not coupled: new insights into the roles of RAD51 and DMC1 in plant meiotic recombination. Plant J. 69: 921-933. Puizina, J., Siroky, J., Mokros, P., Schweizer, D. and Riha, K., 2004 Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11dependent genome fragmentation during meiosis. Plant Cell 16: 1968-1978. Ravi, M., Kwong, P. N., Menorca, R. M., Valencia, J. T., Ramahi, J. S. et al., 2010 The rapidly evolving centromere-specific histone has stringent functional requirements in Arabidopsis thaliana. Genetics 186: 461-471. Richards, E. J., and Ausubel, F. M., 1988, Isolation of a higher eukaryotic telomere from Arabidopsis thaliana. Cell 53: 127-136. Sánchez-Morán E., Armstrong S. J., Santos J. L., Franklin F. C. H., and Jones G. H., 2001 Chiasma formation in Arabidopsis thaliana accession Wassileskija and in two meiotic mutants. Chromosome Res. 9: 121-128. Sánchez-Morán E., Armstrong S. J., Santos J. L., Franklin F. C. H., and Jones G. H., 2002 Variation in chiasma frequency among eight accessions of Arabidopsis thaliana. Genetics 162: 1415-1422. 27 Sasaki, T., Kobayashi, A., Saze, H., and Kakutani, T., 2012 RNAi-independent de novo DNA methylation revealed in Arabidopsis mutants of chromatin remodeling gene DDM1. Plant J. 70: 750-758. Saze, H., MittelstenScheid, O., and Paszkowski, J., 2003 Maintenance of CpG methylation is essential for epigenetic inheritance during plant gametogenesis. Nature Genet. 34: 65-69. Schommer, C., Beven, A., Lawrenson, T., Shaw, P. and Sablowski, R., 2003 AHP2 is required for bivalent formation and for segregation of homologous chromosomes in Arabidopsis meiosis. Plant J. 36: 1-11. Schubert, V., Berr, A., and Meister, A., 2012 Interphase chromatin organisation in Arabidopsis nuclei: constraints versus randomness. Chromosoma 121: 369-387. Singh, M., Goel, S., Meeley, R. B., Dantec, C., Parrinello, H. et al. 2011 Production of viable gametes without meiosis in maize deficient for an ARGONAUTE protein. Plant Cell 23: 443-458. Stein, P., Rozhkov, N. V., Li, F., Cárdenas, F. L., Davydenko, O. et al., 2015. Essential role for endogenous siRNAs during meiosis in mouse oocytes. PLoS Genet. 11: e1005013. Volpe, T., Schramke, V., Hamilton, G. L., White, S. A., Teng, G. et al., 2003 RNA interference is required for normal centromere function in fission yeast. Chromosome Res. 11: 137-146. Wahls, W. P., Siegel, E. R., and Davidson, M. K., 2008 Meiotic recombination hotspots of fission yeast are directed to loci that express non-coding RNA. PLoS One 3: e2887. 28 Wang, Y., Cheng, Z., Huang, J., Shi, Q., Hong, Y. et al., 2012 The DNA replication factor RFC1 is required for interference-sensitive meiotic crossovers in Arabidopsis thaliana. PLoS Genet. 8: e1003039. Wassenegger, M. 2005 The role of the RNAi machinery in heterochromatin formation. Cell 122: 13-16. Wei, W., Ba, Z., Gao, M., Wu, Y., Ma, Y. et al., 2012 A role for small RNAs in DNA double-strand break repair. Cell 149: 101-112. Wei, Y., Yu, L., Bowen, J., Gorovsky, M. A., and Allis, C. D., 1999 Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell 97: 99-109. Wierzbicki, A. T., Haag, J. R., and Pikaard, C. S., 2008 Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135: 635-648. Wierzbicki, A. T., Ream, T. S., Haag, J. R., Pikaard, C. S., 2009 RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nat. Genet. 41: 630-634. Xiao, W., Custard, K. D., Brown, R. C., Lemmon, B. E., Harada, J. J. et al., 2006 DNA methylation is critical for Arabidopsis embryogenesis and seed viability. Plant Cell 18: 805-814. Yang, X., Boateng, K. A., Yuan, L., Wu, S., Baskin, T. I. et al., 2011 The radially swollen 4 separase mutation of Arabidopsis thaliana blocks chromosome disjunction and disrupts the radial microtubule system in meiocytes. PloS One 6: e19459. 29 Yao, Y., Bilichak, A., Golubov, A. and Kovalchuk, I., 2016. Arabidopsis thaliana siRNA biogenesis mutants have the lower frequency of homologous recombination. Plant Signal. Behav. 11: e1151599. Ye, R., Wang, W., Iki, T., Liu, C., Wu, Y. et al., 2012 Cytoplasmic assembly and selective nuclear import of Arabidopsis Argonaute4/siRNA complexes. Mol. Cell 46: 859-870. Ye, R., Chen, Z., Lian, B., Rowley, M. J., Xia, N. et al., 2015. A Dicer-independent route for biogenesis of siRNAs that direct DNA methylation in Arabidopsis. Mol. Cell 61: 222-235 Yelina, N. E., Choi, K., Chelysheva, L., Macaulay, M., De Snoo, B. et al., 2012 Epigenetic remodeling of meiotic crossover frequency in Arabidopsis thaliana DNA methyltransferase mutants. PLoS Genet. 8: e1002844. Zhang, H., and Zhu, J. K., 2011 RNA-directed DNA methylation. Curr. Opin. Plant Biol. 14: 142-147. Zilberman, D., Cao, X., and Jacobsen, S. E., 2003 ARGONAUTE4 control of locusspecific siRNA accumulation and DNA and histone methylation. Science 299: 716-719. Zilberman, D., Cao, X., Johansen, L. K., Xie, Z., Carrington, J. C. et al., 2004 Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. Curr. Biol. 14: 1214-1220. 30 Table 1 Metaphases I with decondensed chromosomes. MUTANT METAPHASES I WITH DECONDENSED CHROMOSOMES (%) n kyp-4 4 137 ago6-2 6 109 kyp6-1 drm1-2 drm2-2 9 66 dcl3-1 22 105 ago4-2 27 177 nrpe1-11 44 71 ago4-1 47 121 n: number of cells analyzed. 31 Table 2 Mean chiasma frequencies per cell, per bivalent and per bivalent arm (short vs. long) in Ler, Col and mutants analyzed in this study. BIVALENTS 1 Ler ago4-1 Col nrpd1a-8 kyp-4 ago6-2 kyp-6 drm1-2 drm2-2 met1-3 drm1-2 drm2-2 dcl3-1 nrpe1-11 cmt3-11 drm1-2 drm2-2 2 3 4 5 s l s l s l s l s l - - 0.56 1.02 0.97 1.00 0.52 0.95 0.86 1.16 2.35 (0.25) 1.57 (0.17) 1.97 (0.21) 1.48 - - 0.62 0.90* 0.74*** 1.05 0.60 2.13* (0.24) 1.52 (0.17) 1.80* (0.20) 1.48 - - 0.61 1.14 0.90 1.26 0.48 2.52 (0.25) 1.75 (0.17) 2.16 (0.21) 1.49 - - 0.78* 1.10 0.97 1.17 0.67* 2.63 (0.25) 1.88 (0.18) 2.13 (0.20) 1.72* - - 0.80* 1.11 0.97 1.13 0.63 2.51 (0.24) 1.91 (0.18) 2.11 (0.20) 1.71* - - 0.80* 1.12 0.86 1.10* 0.74** 2.34 0.23() 1.92 (0.19) 1.96* (0.19) 1.90** - - 0.76 1.13 0.97 1.13 0.52 2.37 (0.23) 1.90 (0.18) 2.18 (0.21) 1.66 - - 0.79* 1.09 0.97 1.21 0.56 2.35 (0.23) 1.88 (0.18) 2.18 (0.21) 1.71 - - 0.80* 1.07 0.92 1.12 0.69** 2.43 (0.24) 1.87 (0.18) 2.04 (0.20) 1.72* - - 0.75 1.15 0.91 1.11* 0.61 2.39 (0.24) 1.91 (0.19) 2.02 (0.20) 1.71* - - 0.62 1.04 0.94 1.10* 0.60 2.42 (0.24) 1.65 (0.17) 2.04 (0.21) 1.62 - - 0.78* 0.97** 0.86 1.04** 0.67* (0.16) 2.02 0.88 0.84 (0.17) 1.91 1.01 0.97 (0.15) 2.28 1.05 1.00 (0.16) 2.32 1.08 1.00 (0.16) 2.31 1.16 1.00 (0.18) 2.22 1.13 0.96 (0.16) 2.24 1.15 (0.21) 1.07 (0.22) 1.30 (0.22) 1.32 (0.22) 1.31 (0.22) 1.22 (0.21) 1.28 (0.22) 1.00 1.12* (0.17) 2.12 (0.21) 1.03 0.96 (0.17) 2.21 1.11 1.25 (0.22) 0.98 1.12** (0.17) 2.11* (0.21) 1.02 0.96 (0.16) 2.15 1.03 0.99 1.19 (0.22) 1.10** C n 9.38 63 8.84** 90 10.20 69 10.68* 60 10.53 75 10.34 50 10.34 67 10.29 34 10.27 75 10.14 66 9.88 52 9.61** 69 2.17*** (0.23) 1.75 (0.18) 1.90** (0.20) 1.70* (0.18) 2.09* (0.22) C: mean cell chiasma frequency; n: number of cells; s: short arm; l: long arm. The values in parentheses are the bivalent chiasma frequencies as proportions of the total cells. *, p < 0.05; **, p < 0.01; *** p < 0.001. Chromosome 1 is considered as a whole because it is not possible to distinguish chromosome arms. ago4-2 32 Figure 1 Meiotic stages in PMCs from Ler, ago4-1, and ago4-2. (A, B, F, J, N, R, V) Representative images from Ler PMCs. (C, D, G, H, K, L, O, P, S, T, W, X) Representative images from ago4-1 PMCs. (E, I, M, Q, U, Y) Representative images from ago4-2 PMCs. (A, C, E) Pachytene. (B) Enlarged centromeric and pericentromeric regions from A. (D) Enlarged centromeric and pericentromeric regions from B. (F-I) Diplotene. (J-M) Metaphase I. (N-Q) Anaphase I. Red arrows indicate a chromosome bridge (O), delayed segregation of homologous chromosomes (P), and lagging chromosomes (Q). (R-U) Metaphase II. Red arrows indicate laggards in S, T and U. (VY) Tetrads. Red arrows indicate chromatin accumulation in W and some micronuclei in X and Y. Bars = 5 µm. (Z) Analysis of chromosome condensation at metaphase I and polyad formation in ago4-1 and ago4-2. Differences were observed in both mutants compared to the respective WT backgrounds. *, p < 5 x 10-2; **, p < 10-2; ***, p < 10-3. 33 Figure 2 FISH to detect centromeres (pAL1) and telomeres (PLT11) at pachytene. (A, D) WT. (B, E) ago4-1. (C, F) ago4-2. Centromeres are showed in green and telomeres in red. White arrows indicate pAL1 signals. Bars = 5 µm. 34 Figure 3 Immunolocalization of 5-methyl cytosine in PMCs from WT and ago4-1 plants. (A, B) Ler. (C, D) Details of A and B. (E, F) ago4-1. (G, H) Details of E and F. Regions of the further enlarged pictures are indicated. White arrows point out centromeres. Bars = 5 µm. 35 Figure 4 Immunolocalization of H3S10Ph in ago4-1. (A-I) Anaphases I. Arrows indicate bridges or chromosome fragments. (J-O) Metaphase II. (P-R) Polyad. Bars = 5 µm. 36 Figure 5 Immunolocalization of α-tubulin and CENH3 in PMCs from Ler and ago4-1. (A-H) WT. (I-T) ago4-1. (A-D, M-P) Anaphase I. The white arrow indicates a microtubule bundle in opposite orientation from the spindle. (E-H, Q-T) Anaphase II. (I-L) Metaphase I. The white arrow indicates a microtubule bundle in opposite orientation from the spindle, depicted by a double-headed arrow. Bars = 5 µm. 37 Figure 6 Cytological analyses of mitosis in somatic cells from ago4-1. (A-D) WT. (E-H) ago4-1. (I-L) ago4-2. (A, E, I) Prophase. (B, F, J) Metaphase. (C, G, K) Anaphase. Arrows indicate a lagging chromatid (G) and an interchromatid bridge (K). (D, H, L) Telophase. Bars = 5 µm. (M) Cells were scored to analyze the decondensation at metaphase, delays in chromatid segregation, and presence of interchromatid bridges at anaphase. There were statistical differences respect to WT in chromosome condensation at metaphase (ago4-1) and anaphase alterations (ago4-1 and ago4-2). *, p < 5 x 10-2; **, p < 10-2; ***, p < 10-3. 38